Vitamin B12 (aka cyanocobalamin) is an essential nutrient to humans but it is only synthesized by prokaryotes. The biologically active form of the vitamin is known as coenzyme B12 (aka adenosylcobalamin, AdoCbl). The conversion of the vitamin to the coenzymic form requires the formation of a unique organometallic bond between the cobalt atom of B12 and the adenosyl group from ATP. We seek to understand the molecular details of the step of the pathway that forms the Co-C bond of the coenzyme. The reaction is very unfavorable but we have substantially improved our understanding of how the energy barrier that opposes the reaction is overcome. We have studied the mechanism of catalysis of the CobA adenosyltrasnferase enzyme of the human pathogen Salmonella enterica. We recently discovered a new type of adenosyltransferase (EutT) in S. enterica that is likely to catalyze the reaction via a mechanism that involves an as-yet-undefined metal center. We propose to continue our mechanistic analysis of CobA and to initiate the biochemical, genetic and structural characterization of the EutT enzyme. We also propose to study two new enzymes of the pathway that are unique to archaea. These enzymes, CobY and CbiS, are involved in the late steps of the assembly of the pathway and in the salvaging of preformed precursors from the environment, respectively. In bacteria, the counterpart for the archaeal CobY enzyme (CobU in S. enterica) is evolutionarily unrelated to CobY, and is more complex. We have a unique opportunity to dissect two different mechanisms of catalysis that must have evolved in response to different selective pressures. A better understanding of the bacterial CobU enzyme is of interest since this enzyme is needed for de novo synthesis of B12 and for salvaging precursors from the environment, hence it represents a potential target for the development of new antibiotics. The CbiS enzyme represents a new pathway for precursor salvaging. What is attractive about CbiS is that it has only been found in archaea living in >100[unreadable]C. CbiS is actually two enzymes in one, and its biochemical, structural and genetic analyses will yield valuable information about thermostability of proteins as well as strategies used by cells occupying extreme environments to stabilize key steps of the pathway. Lastly, we will begin to dissect the biosynthetic pathways for the lower ligand base. One pathway is largely non-enzymatic while the other one is a piecemeal pathway.